The present invention relates to fields using devices and methods for detecting nucleic acid hybridization.
The following is a discussion of relevant art, none of which is admitted to be prior art to the appended claims.
Nucleic acid probe technology has application in detection of infectious disease and genetic and cancer screening. Nucleic acid based probe methods offer several advantages over conventional microbiological or immunological methods for detection of organisms, as described by Nakamura and Bylund (J. Clinical Laboratory Analysis, 6, 73-83, 1992).
Methods to amplify either the number of copies of the nucleic acid available for detection or the signal generated after hybridization of the nucleic acid probe have been utilized. A review of nucleic acid based detection methods and various amplification schemes such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription based amplification, cycling probe reaction, Qxcex2 replicase, and strand displacement amplification may be found in M. J. Wolcott, Clinical Microbiology Reviews, 5, October 1992, pp 370-386.
U.S. Pat. No. 5,175,270 describes an amplification reagent consisting of layers of nucleotide polymers containing double stranded and single stranded sections. Each section has an end which is capable of hybridizing with another molecule.
Probe or hybridization assays are often based on the attachment of an oligonucleotide probe to a surface in order to capture a target nucleic acid molecule (analyte) from a sample. The attachment of this probe to the surface may be through covalent bonds or through a variety of passive absorption mechanisms (e.g., hydrophobic or ionic interactions).
U.S. Pat. No. 5,279,955 describes an immobilization process which uses a heterofunctional cross-linker for a plastic support. The cross-linker consists of a central ring which is hydrophobic and interacts with the plastic, and a hydrophilic chain which terminates in a group capable of reacting with a nucleic acid. Covalent attachment is achieved through a succinyl-olivetol-N-hydroxysuccinimide.
U.S. Pat. No. 5,262,297 describes immobilization of a probe through co-polymers which contain reactive carboxylic acid groups and an 8-50 atom spacer with two or more unsaturated groups within the spacer.
U.S. Pat. No. 5,034,428 describes an immobilization process for probes in which a monomer is joined onto a hydrophilic solid support which can be irradiated in an oxygen free atmosphere. This method provides for covalent attachment of the probe.
U.S. Pat. No. 4,806,546 also describes an immobilization process for an amide modified nylon. The method relies on an amidine linkage under anhydrous conditions in the presence of an alkylating agent.
Maskos and Southern, 20 Nucleic Acids Research 1679, 1992, describe a linker system for the attachment of a nucleic acid to a glass support. The linker system allows for the chemical synthesis of a strand of nucleic acids on the surface. The primary advantage of the linker is that it is stable to an ammonia treatment which is required in the synthesis of the polynucleotide. A hexaethylene glycol spacer is incorporated into the linker which attaches to the glass through a glycidoxypropyl silane which terminates in a primary hydroxy group. The silane is condensed onto silane groups on a solid support. Additional cross-linking may be obtained by introducing water so that the epoxide group is cleaved to a diol. An acidic solution facilitates this process. The length of the linker may be varied by changing the spacer to ethylene glycol, pentaethylene glycol, etc.
Nucleic acid probes that have hybridized to their target sequence are detected based on various methods that introduce a detectable chemiluminscent, fluorescent or other identifiable label into a nucleic acid probe. Several of these techniques are described in U.S. Pat. Nos. 4,968,602, 4,818,680, 5,104,791, and 5,272,056, and International applications WO91/00926 and GB2169403A.
Arnold et al., U.S. Pat. No. 5,283,174 describe the use of a chemiluminscent label with DNA probes. The label is composed of an acridinium ester and has a number of desirable properties. It is stable to hybridization conditions, light is emitted only upon exposure to an alkaline peroxide, the emission kinetics are rapid, and the label on the unhybridized probe can be destroyed without an impact on the signal generated by hybridized probe.
U.S. Pat. No. 5,089,387 describes a diffraction assay for the detection of DNA hybridization. In this invention, a solid support, generally silicon or polysilicon, is coated with a DNA probe. These surfaces are required to inherently adhere the DNA probe to the surface. Once the surface is coated with the probe, the surface is selectively inactivated to provide a series of very strictly controlled reactive probe lines for the generation of the diffraction grating. The unreacted surface is required to be non-light disturbing. The diffraction grating is only generated upon the addition of the analyte to the surface. The angle of diffraction is a function of the wavelength of incident light and the density and spacing of the individual gratings on the surface. A single detector or a multiple detector array may be used to detect and measure the light from all desired orders of the diffracted light.
Mixed phase systems have typically been used to perform hybridization assays. In mixed phase assays the hybridizations are performed on a solid phase such as nylon or nitrocellulose membranes. For example, the assays usually involve loading a membrane with a sample, denaturing the DNA or creating single stranded molecules, fixing the DNA or RNA to the membrane, and saturating the remaining membrane attachment sites with heterologous nucleic acids to prevent the probe reagent from adhering to the membrane in a non-specific manner. All of these steps must be carried out before performing the actual hybridization.
This invention features improved devices, and methods for producing and using optical devices, for detecting the presence or amount of a specific target nucleic acid within a sample. The current invention is based on a probe coated substrate which is optically active. Surfaces can be pre-selected for the type of optical thin film detection to be employed, and enable direct detection of the hybridization reaction through the interaction of light with thin films. Detection of specific target nucleic acid sequences is also referred to as sequence analysis.
This invention also describes materials and methods for producing optically active solid supports or devices for use in nucleic acid hybridization assays and immobilization of nucleic acid probes to such surfaces. These surfaces are compatible with a wide range of optical thin film detection methods, all of which utilize some interaction of thin films with light. Such optical thin film detection methods include optical interference, ellipsometryxe2x80x94comparison, null, photometric and other modifications, attenuation of polarized light at non-Brewster angles, profilometry, scanning tunneling microscopy, surface plasmon resonance, evanescent wave techniques, reflectometry, or atomic force microscopy.
The direct optical thin film detection methods of the current invention are extremely sensitive to changes in mass at the surface of an optically active substrate. These optical thin film detection methods provide increased sensitivity for hybridization assays without the introduction of signal generating labels or pre-assay target amplification with its accompanying complexity and limitations. Thus, assay protocols utilizing optical thin film detection methods are greatly simplified, are more rapid, and less costly than conventional indirect assay methods. Total assay times may vary from one hour to a few minutes from the initiation of the assay protocol (i.e., from the time that the target nucleic acid containing sample is contacted with the device). Such devices also allow for assay results to be visualized as a color change or detected by instrumented formats.
The methods and devices of the claimed invention by concentrating on improving characteristics of the surface immobilized probe and detection of the hybridization reaction without the use of a labelled component allow for rapid, convenient, and sensitive assays for diagnostic (routine clinical) and research use.
In a first aspect the invention features an optical assay device for detecting the presence or amount of a target nucleic acid. The device includes an optically active substrate capable of producing a thin film effect, and exhibiting a first set of reflective and transmissive properties in response to light impinging thereon, and exhibiting a second set of reflective and transmissive properties different from said first set, in response to light when the target nucleic acid is bound to a target specific capture probe and creates a change in mass on said optically active substrate. The target specific capture probe is attached to an attachment layer that is present on the optically active layer. Detection of the second set of reflective or transmissive properties indicates the presence or amount of the target nucleic acid.
An xe2x80x9coptically active substratexe2x80x9d comprises an optically active surface. Such a substrate may consist of more than one layer (multi-layered), for example; base material, a conducting metal layer of aluminum, chromium, or a transparent conducting oxide, and a layer of amorphous silicon, wherein the metal layer is positioned adjacent the amorphous silicon. Alternatively, the multi-layered substrate may comprise a layer of base material (any solid material on which optically active layers may be applied), and a layer of amorphous silicon adjacent the base material. The base material is selected from any of the group consisting of glass, fused silica, plastics, semiconductors, ceramics, and metals, and may be either rigid or flexible. The optically active surface substrate also serves as a solid support for the nucleic acid target probe and any amplification components (if necessary) and must be chemically stable to the application of the attachment layer, the nucleic acid probe, and all assay reagents. The properties of the optically active substrate are matched to the direct optical thin film detection method employed.
An xe2x80x9coptically active surfacexe2x80x9d is a surface that participates in the generation of an optical effect such that the light impinging upon that surface is in some way altered. Such optically active surfaces may be adapted to respond not only to polychromatic light (e.g., white light) but also to monochromatic light (e.g., laser light, which may be inherently polarized). The optically active surface may be designed to reflect or transmit light. The substrate may inherently possess an optically active surface or the substrate may require an optically active surface be applied to introduce the desired optical characteristics. Thus, the optically active substrate may inherently possess the properties required for a given thin film detection method, or a surface may be modified to provide the required optical properties. Materials which are suitable for optically active substrates include monocrystalline or polycrystalline silicon, glasses, ceramics, metals, amorphous silicon on glass, amorphous silicon on plastic, plastics, and composites of these materials.
By xe2x80x9cthin film effectxe2x80x9d is meant that light impinging on an optically active surface or substrate is attenuated or modulated in its reflective or transmissive properties.
The first set of reflective or transmissive properties is defined as a combination of wavelengths of light, or a spectral distribution, or an intensity of one or more of wavelengths, or a degree or amount of elliptical polarization, or an amount of polarization rotation. The substrate also exhibits a second set of reflective or transmissive properties which is different from the first set. The second set of reflective or transmissive properties is exhibited in response to the same light when the target nucleic acid is present on the surface. This second set of reflective or transmissive properties is due to a change in mass on the optically active substrate. The change from one set of properties to another can be measured either by use of an instrument, or by eye. The optical active substrate is selected to be compatible with the method of optical thin film detection.
The target nucleic acid is selected so that it specifically identifies a single organism or gene. The target sequence is selected such that the stringency of the assay conditions will promote or enhance the specificity of the assay. The target nucleic acid may be a DNA or RNA (rRNA, tRNA, mRNA, small nuclear RNA""s (SNURPS)) molecule either intact or a fragment thereof covering a range of molecular weights. Fragments may be generated enzymatically, chemically, or by mechanical shearing. The target may be free or contained within a larger complex, i.e., complexed with protein(s). The target may be single or double stranded oligonucleotide and the source may be a bacterium, a virus, a human cell, and may be isolated from a culture medium or a biological fluid.
A xe2x80x9ctarget specific capture probexe2x80x9d refers to a synthetic or biologically produced nucleic acid which by design contains specific, complimentary nucleotide sequences that allow it to hybridize to a target nucleic acid sequence. The probe may be composed of deoxyribose, ribose, or a combination of these nucleotides (chimera) and may be synthesized chemically, isolated from a biological source, or cloned. It may be a linear strand or may contain branch points to increase the density of immobilized probe. The probe may provide a single or multiple copies of the sequence complimentary to the target nucleic acid. The nucleic acid probe or capture probe is selected to specifically hybridize a target nucleic acid. When more than one probe is used in a hybridization assay, each probe should recognize a unique sequence well separated from each other within the target oligonucleotide.
Hybridization is the process by which two partially or completely complementary strands of nucleic acid are allowed to come together, under predetermined reaction conditions, in an antiparallel fashion to form double stranded nucleic acid with specific and stable hydrogen bonds. The nucleic acid capture probe sequence is selected to specifically interact with the target molecule or analyte at a pre-determined degree of stringency. The probe length is pre-determined to provide the required specificity at the degree of stringency used in the assay. Assay conditions are set so that the stringency of hybridization between the capture probe and the target nucleic acid provides the required specificity.
Stringency of a particular set of hybridization conditions is defined by the length and base composition of the probe/target duplex as well as by the amount and geometry of mispairing between the two nucleic acids. It is governed by such solution parameters as concentration and type of ionic species, type and concentration of denaturing agents, precipitating agents, and the temperature of hybridization. As stringency increases, longer probes are preferred for the generation of stable hybrids. The stringency of the reaction conditions control the specificity of a hybridization assay. For a complete review of hybridization assay conditions see xe2x80x9cMolecular Cloningxe2x80x94A Laboratory Manualxe2x80x9d, second edition, Sambrook, Fritisch, and Maniatis. Also, software has been developed that can more accurately predict hybridization conditions than these traditional methods.
The capture probe may be modified to promote passive adhesion to the attachment layer or be chemically reactive with other surface immobilized materials to allow covalent attachment. The modifications should be made to the sugar-phosphate backbone to prevent stearic problems associated with attachment at the bases. A linker may be introduced into the probe sequence to space the probe from the surface and facilitate attachment.
It is critical that the probe on the surface be attached so that the individual monomers are free to hybridize with the target without stearic inhibition. This may be particularly relevant when simple hydrophobic interactions are used to immobilize the probe because base interactions will be blocked. Covalent methods of attachment can eliminate these considerations and also improve the efficiency and kinetics of hybridization. A determination of the correct position for probe attachment (horizontal or end) may be made empirically. In addition, covalent attachment of the probe should not block additional binding sites on the surface. Thus, the probe surface density will not be reduced. Covalent attachment techniques which modify any or all of the probe nucleotide residues must be avoided as they could impact subsequent hybridization reactions.
By xe2x80x9cmass changexe2x80x9d is meant a modification (increase or decrease) in the amount of material at the surface of an optically active substrate such that one or more of the reflective and/or transmissive properties of the optically active substrate are altered.
In many cases the optically active substrate selected in conjunction with a specific optical detector is not readily reactive nor will it retain the nucleic acid probe. Thus, the surface must be modified with a material which enhances or assists in the immobilization of the probe onto the optical substrate. These materials are referred to as attachment layers. The materials selected for the attachment layer must provide the following characteristics.
Attachment layers must react either in a covalent or very strong non-covalent manner with the optical substrate. The probe may be adhered to the attachment layer either covalently or passively. The attachment layer may be activated to react with the probe, the probe may be activated to react with the attachment layer, or both may be chemically activated to react with each other. The attachment layer can be selected to determine the type of interaction (hydrophobic, hydrophilic, ionic, hydrogen bonded) which occurs between itself and the capture probe. Attachment layers may be applied to the surface of the optical substrate by spin coating, solution coating, vapor deposition, spray coating or the like. The layer must be applied in a uniform fashion from a solvent system which will not damage the optical substrate or the chemical properties of the attachment layer. Ideally, the solvent will volatilize from the surface film during the final curing process.
In a preferred embodiment the invention features a device with an amplifying probe reagent able to bind to the target nucleic acid and create an increase in mass change on the optically active layer, without disrupting the thin film effect, when the target nucleic acid is bound to the target specific capture probe.
An xe2x80x9camplifying probe reagentxe2x80x9d is designed to increase the mass of the immobilized target nucleic acid on the optically active substrate. This amplification may be required to increase the sensitivity of the hybridization assay in the optical thin film detection system. The amplifying probes are attached to another catalytic component or particle which will increase the mass when the target nucleic acid is captured on the surface. The only requirement for such a material is that it can be tightly associated with the amplifying probe and that the resulting mass deposited at the surface not disrupt the thin film effect. When a catalytic component is attached to the signal amplifying probe, or amplifying probe, it may act on yet another material, further increasing mass on the optical substrate. There may be a surface immobilized capture probe and one or more signal amplifying probes.
By xe2x80x9cincrease in massxe2x80x9d is meant that the mass change produced is greater than that produced by the binding of the target nucleic acid.
By xe2x80x9cwithout disrupting the thin film effectxe2x80x9d is meant that the optical effect generated by light impinging on the optically active substrate is not destroyed by the introduction of additional materials to the surface of the substrate. For example, materials which would scatter light are disruptive to a thin film interference or other reflective techniques, as they would artificially reduce the signal available to a detector.
In further preferred embodiments the amplifying probe reagent comprises a target specific nucleic acid sequence and a catalytic component or a particle; the catalytic component is capable of interacting with additional materials to increase the mass change and result in a second set reflective and transmissive properties without disrupting the thin film effect; the catalytic component is an enzyme; and the particle is selected from the group consisting of metallic particles, silica particles, and film forming latexes.
By xe2x80x9ccatalytic componentxe2x80x9d is meant any material which can interact with a substrate to create an insoluble product. Examples of these materials include enzymes, metals and other components which cause a reaction to occur faster than it would occur in the absence of that material.
By xe2x80x9cparticlexe2x80x9d is meant a material such as a film forming latex or small non-scattering metal or glass particles.
By xe2x80x9cadditional materialsxe2x80x9d is meant an assay component which is specifically modified by the catalytic component to produce a precipitating material. The precipitating agent may be a substrate for an enzyme, e.g., containing 3,3xe2x80x2,5,5xe2x80x2-tetramethylbenzidene when the enzyme conjugate has an immobilized peroxidase or the precipitating agent is a substrate containing 5-bromo-4-chloro-3-indolyl phosphate when the conjugate is alkaline phosphatase.
The following preferred embodiments are applicable to the device with and without the use of an amplifying probe reagent. The attachment layer is selected from the group consisting of polymeric siloxanes, mixtures of polymeric siloxanes, film forming latexes, and silyl modified nucleotides.
The optically active substrate is selected from the group consisting of silicon, glass, amorphous silicon, plastic, metals, amorphous silicon on glass, amorphous silicon on plastic, and composites of these materials.
An anti-reflective film may be provided between the optically active substrate and the attachment layer. This film which may be formed from silicon nitride, silicon oxides, titanium dioxide, silicon oxynitride or cadmium sulfide and the like. This film acts to cause incident light to undergo interference such that a specific color is produced on the surface of the substrate. This film interacts with other layers on the substrate to ensure that a color change or wavelength intensity change is observed when the target nucleic acid is present on the device.
In a preferred embodiment the anti-reflective film is selected from the group consisting of silicon nitride, composites of silicon and silicon oxides, ti tanium dioxide, titanates, silicon carbide, diamond, and cadmium sulfide.
In a further preferred embodiment detection is by an optical thin film detection method selected from the group consisting of ellipsometry, optical interference, attenuation of polarized light at non-Brewster angles, profilometry, reflectometry, scanning tunneling microscopy, atomic force microscopy, surface plasmon resonance, evanescent wave techniques, interference spectroscopy, and various other forms or combinations of polarimetry, reflectometry, spectroscopy, and microscopy. This invention concerns the application of such technologies for the direct detection or measurement of changes in the thickness, density, refractive index, optical thickness, or mass of thin films resulting from the concentration-dependent immobilization of target nucleic acid on a surface coated with a suitably selected probe. All these properties maybe applicable when a thickness or mass change occurs. Such thin film assay technologies directly detect or quantitate the material of interest, and are alternatives to conventional solid phase assays.
In other preferred embodiments the first and second set of reflective or transmissive properties are visual interference colors; the first and second set of reflective or transmissive properties are changes in the degree of rotation observed in polarized light; the first and second set of reflective or transmissive properties are changes in the ellipticity of the impinging polarized light.
By xe2x80x9cvisual interference colorsxe2x80x9d is meant the change in interference colors produced by modifying an anti-reflective layer.
By xe2x80x9cdegree of rotation observed in polarized lightxe2x80x9d is meant the attenuation of the degree or amount of rotation measured in polarized light incident upon the optically active substrate before and after reaction with the target molecule as measured by a change in intensity of the incident light.
By xe2x80x9cchanges in ellipticity of the impinging polarized lightxe2x80x9d is meant the attenuation of the ellipticity measured in polarized light incident upon the optically active substrate before and after reaction with the target molecule as measured by a change in intensity of the incident light.
In additional preferred embodiments the target nucleic acid is DNA, rRNA, mRNA, tRNA, small nuclear RNA, or complexes of said nucleic acid with other materials; the target nucleic acid is single stranded or double stranded; the target nucleic acid is obtained from a bacterium, a virus, a human cell, a culture, serum, plasma, blood, urine, sputum, tissue sample, or other biological fluid. Target nucleic acid is obtained from a source by the use of routine extraction and isolation procedures familiar to those who practice the art. The target nucleic acid may also require denaturation to allow for hybridization with the capture probe, such procedures are also standard in the art.
In a further preferred embodiment, the capture probe is a synthetic or biologically produced nucleic acid which may consist of DNA, RNA, or a chimera; the capture probe is a linear strand providing single or multiple copies of a sequence complimentary to the target nucleic acid; the capture probe is a branched, multi-copy sequence complimentary to the target nucleic acid; the capture probe is adhered to the attachment layer by hydrophobic interactions; the capture probe is adhered to the attachment layer by hydrophilic interactions; the capture probe is adhered to the attachment layer by ionic interactions; the capture probe is adhered to the attachment layer by hydrogen bonding; the attachment layer comprises a film forming latex and the capture probe is a chimeric probe which is covalently attached to the attachment layer.
By xe2x80x9cchimeraxe2x80x9d is meant that the capture probe consists of both DNA and RNA. For example, a single ribonucleotide maybe incorporated into a DNA capture probe and is used in covalent attachment of the capture probe to the optically active surface. The ribonucleotide residue may be treated with an oxidizing agent such as periodate to produce a dialdehyde which is reactive with a carbonyl hydrazide modified surface. A covalent modification of the optically active substrate is produced.
By xe2x80x9cbranched multi-copy sequencexe2x80x9d is meant a probe with a branch point such that from emanating branches a copy of the specific capture sequence is extended, thus presenting multiple copies of the capture probe.
In a second embodiment the invention features a method for detecting the presence or amount of a target nucleic acid. The method consists of: preparing a sample, potentially containing a target nucleic acid, for hybridization, contacting the sample with a capture probe supported on an optically active substrate under conditions such that the probe specifically hybridizes to the target, and determining the amount or presence of the target on the optically active substrate by an optical thin film detection method.
By xe2x80x9cpreparing a samplexe2x80x9d is meant treatment of a sample so that a target nucleic acid is in a condition that would allow for hybridization with a capture probe and includes extraction and isolation procedures known to those skilled in the art.
In a preferred embodiment the target nucleic acid after being bound to the capture probe is contacted with an amplifying probe reagent.
In a third embodiment, the invention features a method for increasing capture probe density whereby the probe is applied to an attachment layer on an optically active substrate by spin coating. The density of the immobilized probe is increased by this process relative to a conventional solution coating process.
By xe2x80x9ccapture probe densityxe2x80x9d is meant the amount of capture probe available to bind the target nucleic acid or the number of capture probes per specific amount of surface area.
By xe2x80x9cspin coatingxe2x80x9d is meant the standard semiconductor coating procedure.
In a fourth aspect, the invention features a kit for an optical assay for a target nucleic acid having a test device with an optically active substrate with an attached capture probe which is reactive with the target nucleic acid, and a reagent adapted to react with the target bound to the surface to alter the mass on the surface. Preferably, the reagent is an enzyme conjugate or a polymeric film forming latex.
The kit comprises an optically active substrate coated with a target specific capture probe. The kit may contain reagents for the extraction of the target oligonucleotide from a biological sample, reagents to destabilize the initial target duplex or other secondary structure, hybridization reagents, stringency washes, sample processing tubes and transfer pipettes, and amplifying reagents if required.
In a fifth embodiment the invention features an optical assay device for detecting the presence or amount of a target nucleic acid comprising: an optically active substrate exhibiting a first set of reflective properties in response to light impinging thereon, and exhibiting a second set of reflective properties different from the first set, in response to the light when the target nucleic acid is bound to a target specific capture probe so as to result in a mass change on the optically active substrate. The target specific capture probe attached to said optically active substrate, and detection of said second set of reflective properties indicates the presence or amount of said target nucleic acid.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.